Site-targeted transformation using amplification vectors

ABSTRACT

A process of causing a targeted integration of DNA of interest into a plant cell nuclear genome, comprising; i) providing plant cells with an amplification vector, or a precursor thereof, capable of autonomous replication in plant cells, said vector comprising; a) DNA sequence(s) encoding an origin of replication functional in plant cells, b) DNA sequence(s) necessary for site-specific and/or homologous recombination between the vector and a host nuclear DNA, and c) optionally, further DNA of interest; ii) optionally providing conditions that facilitate vector amplification and/or cell to cell movement and/or site-specific and/or homologous recombination, and iii) selecting cells having undergone recombination at a predetermined site in the plant nuclear DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 35 U.S.C. §371 National Phase Applicationof International Application Serial No. PCT/EP02/03266, filed Mar. 22,2002 and published in English as PCT Publication No. WO 02/077246 onOct. 3, 2002, which claims priority to German Patent Application SerialNo. 101 14 209.9, filed Mar. 23, 2001, the disclosures of each of whichare incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the genetic modification of plants.Particularly, it relates to a process of site-targeted integration ofDNA into a plant cell nuclear genome. It further relates to vectors forsuch a process and to plant cells, seeds and plants produced thereby.Also, a kit-of-parts is provided for performing the process of theinvention.

BACKGROUND OF THE INVENTION

With current levels of research in the field of plant molecular geneticsand functional genomics, plant transformation is likely to become anincreasingly important tool for plant improvement. Limitations ofcurrent transformation procedures are numerous but one most importantdeficiency of currently used techniques is that they result in randominsertions of target genes in host genomes, leading to uncontrolleddelivery and unpredictable levels of transgene expression. As a result,existing methods require many independent transgenic plants to begenerated and analyzed for several generations in order to find thosewith the desired level or pattern of expression. The vectors for suchnon-targeted transformation must necessarily contain full expressionunits, as the subsequent transformation to the same site is impossible,thus limiting engineering capability of the process. A number ofdifferent approaches have been investigated in an attempt to developprotocols for efficient targeting of DNA at specific sites in thegenome. These efforts include:

-   -   (i) attempts to improve the process of homologous recombination        (that relies on the endogenous cellular recombination machinery)        by over-expressing some of the enzymes involved in        recombination/repair;    -   (ii) attempts to decrease non-targeted recombination by        down-regulating enzymes that contribute to non-specific        recombination;    -   (iii) use of heterologous recombinases of microbial origin;    -   (iv) development of chimeraplasty for targeted DNA modification        in plants.

A brief description of these efforts is summarized below.

Homologous recombination occurs readily in bacteria and yeast, where itis used for gene replacement experiments. More recently it has beendeveloped as a tool for gene replacement in mammals (Mansour et al,1988, Nature, 336, 348-336; Thomas et al, 1986, Cell, 44, 419-428;Thomas et al, 1987, Cell, 51, 503-512), and the moss Physcomytrellapatens (Schaefer & Zryd, 1997, Plant J., 11, 1195-1206). However, it isinefficient in plants. Targeted DNA modification by homologousrecombination is accomplished by introducing into cells linear DNAmolecules that share regions of homology with the target site.Homologous recombination occurs as a result of a repair mechanisminduced by the double-strand breaks at the ends of the DNA fragment.Unfortunately, a competing repair mechanism called non-homologousend-joining (NHEJ) also takes place at a much higher frequency in manyorganisms and/or cell types, rendering selection of the desiredsite-targeted events difficult (Haber, 2000, Curr. Op. Cell. Biol., 12,286-292; Haber, 2000, TIG, 16, 259-264; Mengiste & Paszkowski, 1999,Bio.l Chem., 380, 749-758). In higher plants only a few cases ofsuccessful targeted transformation by homologous recombination have beenreported, and all were obtained with efficiencies of targeted eventsover non-targeted events in the range of 10⁻³ to 10⁻⁵ (Paszkowski etal., 1988, EMBO J., 7, 4021-4026; Lee et al., 1990, Plant Cell; 2,415-425; Miao & Lam, 1995, Plant J., 7, 359-365; Offringa et al., 1990,EMBO J., 9, 3077-3084; Kempin et al., 1997, Nature, 389, 802-803). Thismeans that the screening procedure will involve a very large number ofplants and will be very costly in terms of time and money; in many casesthis will be a futile effort.

Attempts to increase homologous recombination frequencies have beenmade. Investigators have over-expressed some of the enzymes involved indouble-strand break repair. For example, over-expression of either theE. coli RecA (Reiss et al., 1996, Proc Natl Acad Sci USA., 93,3094-3098) or the E. coli RuvC (Shalev et al., 1999, Proc Natl Acad SciUSA., 96, 7398-402) proteins in tobacco has been tried. However, thishas only led to an increase of intrachromosomal homologous recombination(of approximately 10 fold). There was no increase of gene targeting(Reiss et al., 2000, Proc Natl Acad Sci USA., 97, 3358-3363.). Usinganother approach to increase homologous recombination, investigatorshave induced double-strand breaks at engineered sites of the genomeusing rare cutting endonucleases such as the yeast HO endonuclease(Chiurazzi et al, 1996, Plant Cell, 8, 2057-2066; Leung et al., 1997,Proc. Natl. Acad. Sci., 94, 6851-6856) or the yeast I-Sce I endonuclease(Puchta et al., 1996, Proc. Natl. Acad. Sci., 93, 5055-5060). Sitetargeted frequency of 2×10⁻³ to 18×10⁻³ was obtained using the I-Sce Iendonuclease. Although an improvement, this is still inefficient. Inaddition, many of the targeted events contained unwanted mutations oroccurred by homologous recombination at one end of the break only.Incidentally, there is an interesting recent publication describing ahyperrecombinogenic tobacco mutant demonstrating three orders ofmagnitude increase of mitotic recombination between homologouschromosomes, but the gene(s) involved has not been identified yet(Gorbunova et al., 2000, Plant J., 24, 601-611) and targetedrecombination is not involved.

An alternative approach consists of decreasing the activity of enzymes(e.g. Ku70) involved in non-homologous end joining (U.S. Pat. No.6,180,850) to increase the ratio of homologous/non-homologousrecombination events. This approach has been far from being practicallyuseful.

A recently developed approach called chimeraplasty consists of usingDNA/RNA oligonucleotides to introduce single-nucleotide mutations intarget genes. This approach is highly efficient in mammalian cells (Yoonet al., 1996, Proc. Natl. Acad. Sci. USA., 93, 2071-2076; Kren et al.,1999, Proc. Natl. Acad. Sci. USA., 96, 10349-10354; Bartlett et al.,2000, Nature Biotech., 18, 615-622) with a success rate of more than40%. Unfortunately, the efficiency is much lower in plants (Zhu et al.,1999, Proc. Natl. Acad. Sci. USA., 96, 8768-8773; Beetham et al., 1999,Proc. Natl. Acad. Sci. USA., 96, 8774-8778; Zhu et al., 2000, NatureBiotech., 18, 555-558; WO9925853) and reaches only a frequency of10⁻⁵-10⁻⁷. A further severe drawback of using the chimeraplasty approachin plant systems is that it is limited to the introduction ofsingle-nucleotide mutations and to the special case where the introducedmutation results in a selectable phenotype.

Another approach has been to use heterologous site-specific recombinasesof microbial origin. When these recombinases are used, specificrecombination sites have to be included on each side not only of the DNAsequence to be targeted, but also of the target site. So far, this hasbeen a severely limiting condition which gives this approach littlepractical usefulness. Examples of such systems include the Cre-Loxsystem from bacteriophage P1 (Austin et al., 1981, Cell, 25, 729-736),the Flp-Frt system from Saccharomyces cerevisiae (Broach et al., 1982,Cell, 29, 227-234), the R-RS system from Zygosaccharomyces rouxii (Arakiet al., 1985, J. Mol. Biol., 182, 191-203) and the integrase from theStreptomyces phage PhiC31 (Thorpe & Smith, 1998, Proc. Natl. Acad. Sci.,95, 5505-5510; Groth et al., 2000, Proc. Natl. Acad. Sci., 97,5995-6000). Wild-type Lox sites (LoxP sites) consist of 13 bp invertedrepeats flanking an 8 bp asymetrical core. The asymmetry of the coreregion confers directionality to the site. Recombination between LoxPsites is a reversible reaction that can lead to deletions, insertions,or translocations depending on the location and orientation of the Loxsites. In plants, the Cre-Lox system has been used to create deletions(Bayley et al, 1992, Plant Mol. Biol., 18, 353-361), inversions(Medberry et al., 1995, Nucl. Acids. Res., 23, 485-490), translocations(Qin et al., 1994, Proc. Natl. Aced. Sci., 91, 1706-1710); Vergunst etal, 2000, Chromosoma, 109, 287-297), insertion of a circular DNA into aplant chromosome (Albert et al., 1995, Plant J., 7, 649-659),interspecies translocation of a chromosome arm (Heather et al., 2000,Plant J., 23, 715-722), and removal of selection genes aftertransformation (Dale & Ow, 1991, Proc. Natl. Acad. Sc., 88, 10558-62;Zuo et al., 2001, Nat Biotechnol., 19, 157-161). One problem encounteredwhen the Cre-Lox system (or a similar recombination system) is used fortargeted transformation is that insertion of DNA can be followed byexcision. In fact, because the insertion of DNA is a bimolecularreaction while excision requires recombination of sites on a singlemolecule, excision occurs at a much higher efficiency than insertion. Anumber of approaches have been devised to counter this problem includingtransient Cre expression, displacement of the Cre coding sequence byinsertion leading to its inactivation, and the use of mutant sites(Albert et al., 1995, Plant J., 7, 649-659; Vergunst et al., 1998, PlantMol. Biol., 38, 393-406; U.S. Pat. No. 6,187,994). Some site-specificrecombinases such as the Streptomyces phage PhiC31 integrase should notsuffer from the same problem, theoretically, as recombination events areirreversible (the reverse reaction is carried out by different enzymes)(Thorpe & Smith, 1998, Proc. Natl. Acad. Sci., 95, 5505-5510), but theyare limited to animals), but the use of this recombination system inplant cells has not been confirmed yet. There are other flaws thatrender the site-specific recombination systems practically unattractive.First, one needs to engineer a landing or docking site in therecipient's genome, a procedure that is currently done by randominsertion of recombination sites into a plant genome. This eliminatesmost of benefits of the site-specific integration. Second, the frequencyof desired events is still very low, especially in economicallyimportant crops, thus limiting its use to tobacco and Arabidopsis.Expression of recombinant enzymes in plant cells leads to a toxicityproblems, an issue that cannot be circumvented with commonly usedsystems such as Cre-lox or Flp-frt.

WO 99/25855 and corresponding intermediate U.S. Pat. No. 6,300,545disclose a method of mobilizing viral replicons from anAgrobacterium-delivered T-DNA by site-specific recombination-mediatedexcision for obtaining a high copy number of a viral replicon in a plantcell. It is speculated that said high copy number is useful forsite-targeted integration of DNA of interest into a plant chromosomeusing site-specific recombination. However, the disclosure does notcontain information on how to test this speculation. The examples givenin the disclosure do not relate to site-targeted integration. Moreover,the examples cannot provide cells having undergone site-targetedintegration, but only plants showing signs of viral infection such asappearance of yellow spots and stripes at the base of new leavesindicative of the decay of the infected cells. Therefore, the teachingof these references is limited to the infection of cells leading to thedestruction of the cell by the viral vector. The teaching of thesereferences neither allows the determination as to whether or notintegration into the nuclear genome has taken place, let alone theselection of successful site-targeted integration events. This isunderlined by the fact that the references do not contain a disclosureof selection methods for recovering site-targeted transformants.Selection and recovery of transgenic progeny cells containing said DNAof interest site-specifically integrated into the nuclear genome issimply impossible based on the teaching of these references. Moreover,WO 99/25855 and U.S. Pat. No. 6,300,545 are silent on this problem.Further, these documents are silent on homologous recombination.Moreover, the method is limited to replicon delivery by way ofAgrobacterium.

Therefore, it is the problem of the invention to provide a process fortargeted transformation of plants which is sufficiently efficient forpractical purposes.

It is a further problem of the invention to provide a method of targetedintegration of DNA of interest into a plant cell nuclear genome thatallows recovery of integration events, i.e. selection of cells havingundergone recombination in the plant nuclear DNA.

It is a further problem of the invention to provide a method of targetedintegration of DNA of interest into a plant cell nuclear genome byhomologous recombination.

It is therefore a further problem of the invention to provide a methodof targeted integration of DNA of interest into a plant cell nucleargenome by delivery methods independent from Agrobacterium-mediatedmethods.

SUMMARY OF THE INVENTION

This problem is solved by a process of causing a targeted integration ofDNA of interest into a plant cell nuclear genome, comprising:

-   (i) providing plant cells with an amplification vector, or a    precursor thereof, capable of autonomous replication in plant cells,    said vector comprising:    -   (a) DNA sequences encoding an origin of replication functional        in plant cells,    -   (b) DNA sequence(s) necessary for site-specific and/or        homologous recombination between the amplification vector and a        host nuclear DNA, and    -   (c) optionally, a further DNA of interest;-   (ii) optionally providing conditions that facilitate vector    amplification and/or cell to cell movement and/or site-specific    and/or homologous recombination, and-   (iii) selecting cells having undergone recombination at a    predetermined site in the plant nuclear DNA.

Further, a process of causing a targeted integration of DNA of interestinto a plant cell nuclear genome is provided, comprising the followingsteps:

-   (i) transfecting or transforming a plant cell with a first DNA    comprising a sequence which, when integrated in the plant cell    genome, provides a target site for site-specific and/or homologous    recombination;-   (ii) selecting a cell which contains said target site for    site-specific and/or homologous recombination in its nuclear genome;-   (iii) transfecting or transforming said selected cell with a second    DNA comprising a region for recombination with said target site and    a first sequence of interest;-   (iv) optionally providing enzymes for recombination; and-   (v) selecting cells which contain the sequence of interest from the    second DNA integrated at the target site,    whereby at least one of said first or said second DNA is delivered    by an amplification vector, or a precursor thereof, capable of    autonomous replication in a plant cell and comprising DNA sequences    encoding an origin of replication functional in the plant cell.

Further, this invention provides plant cells, seeds and plants obtainedor obtainable by performing these processes and a vector (amplificationvector) or pro-vector (precursor) for performing these processes.Moreover, the invention provides Agrobacterium cells and packaged viralparticles containing said vector or pro-vector.

Finally, the invention provides a kit-of-parts comprising

-   (i) plant cells, seeds or plants, notably according to steps (i)    and (ii) of the above five-step process and-   (ii) a vector or pro-vector according to the invention and/or said    Agrobacterium cells and/or said packaged viral particles.

A further kit-of-parts is provided comprising a vector or a pro-vectorfor performing steps (i) and (ii) of the above five-step process and avector for performing steps (iii) and (iv) of that process.

It has been found that surprisingly the efficiency of site-targetedtransformation of plant cells can be greatly improved by providing DNAsequences for site-specific and/or homologous recombination by anamplification vector. The exact reasons for this improvement are not yetknown but it may be due to an increase of the copy number of thesequence(s) to be targeted. Examples are provided which demonstrate astrong increase of site-targeted insertion events by using amplificationvectors as opposed to non-amplifying vectors. It is even more surprisingthat this increased copy number does not at the same time increase thefrequency of non-targeted or random insertion of the sequence(s) to betargeted into the nuclear genome. As a result, the ratio of targeted torandom insertion frequencies is highly increased by the processes ofthis invention. Most importantly, targeted transformation reaches alevel of efficiency such that it may now become a routine method inplant biotechnology.

Replication of the amplification vector, however, renders selection ofintegration events difficult or impossible since high copy numbers of anamplification vectors lead to disease symptoms, impediment of celldivision and ultimately to death of affected cells. Consequently,progeny cells containing DNA of interest integrated into the nucleargenome cannot be obtained. The inventors were therefore faced with thefollowing dilemma: on the one hand, efficient site-targeted integrationrequires replication of the vector. On the other hand, said replicationprevents selection of cells having undergone recombination in the plantnuclear DNA.

The inventors of the invention have surprisingly identified ways out ofthis dilemma. Preferably, the processes of the invention are designedsuch that the replication of said amplification vector in cellstransformed or transfected with said amplification vector is transient.Transient replication means temporal replication, i.e. a replicationthat lasts for a limited period of time necessary to achieve or todetect homologous and/or site-specific recombination within said cellsand integration of said DNA of interest into the nuclear genome.Transient replication of the amplification vector does preferably notprevent the ability of said cells to divide such that progeny cells areformed which can be selected. Preferably, the amplification vectordisappears in progeny cells. Below, examples are provided whichdemonstrate successful selection of progeny cells according to theinvention.

Transient replication of the amplification vector may be achieved inseveral ways. One possibility is to provide the nucleic acid polymerase(replicase) involved in replicating the amplification vector transientlysuch that replication stops when said polymerase disappears. This may bedone by providing the replicase gene to the plant cell on anon-replicating vector (cf. example 6). Preferably, selection pressureused for maintaining said non-replicating vector may be relieved to thisend. Further, replication may stop or diminish as a result of therecombination event (cf. example 13), e.g. by rendering the replicasegene non-expressible.

The invention further provides a process of causing targeted integrationof DNA of interest into a plant cell nuclear genome comprising:

-   (i) providing plant cells with an amplification vector, or a    precursor thereof, capable of autonomous replication in plant cells,    said vector comprising:    -   (a) DNA sequences encoding an origin of replication functional        in plant cells,    -   (b) DNA sequence(s) necessary for homologous recombination        between the amplification vector and a host nuclear DNA, and    -   (c) optionally, a further DNA of interest;-   (ii) optionally providing conditions that facilitate vector    amplification and/or cell to cell movement and/or site-specific    and/or homologous recombination, and-   (iii) selecting cells having undergone recombination at a    predetermined site in the plant nuclear DNA.

In order to amplify in a plant cell, the amplification vector used inthis invention has to have an origin of replication functional in aplant cell. The origin of replication may be derived from a plantnuclear genome, e.g. from a ribosomal DNA intergenic spacer region.Alternatively, the origin of replication may be of non-plant origin orof synthetic nature. Preferably, the origin of replication is derivedfrom a plant virus, most preferably from a plant DNA virus. The originof replication is functional in a plant cell if it is recognised by areplication enzyme (DNA or RNA polymerase) in said cell. The replicationenzyme is preferably of the same origin as the origin of replication. Ifthe replication enzyme originates from the plant species to betransformed, no foreign replication enzyme has to be provided to saidplant cells. In order to facilitate vector amplification, a replicationenzyme may be provided, notably if said origin of replication originatesfrom a source different from said plant cells. This enzyme may beencoded on the amplification vector, on an additional vector or it maybe incorporated into the plant nuclear genome.

The amplification vector may be a plant virus-derived vector. It may bederived from an RNA virus. In this case it is preferably a DNA copy or areplication intermediate of an RNA virus-derived vector. Preferablyhowever, the vector is derived from a DNA virus. A vector may beconsidered to be derived from an RNA or DNA virus, if it contains atleast one functional element of such a virus. Preferably, such afunctional element is an origin of replication which is recognized by areplication enzyme (polymerase) of that virus.

Geminiviridae are particularly well-suited for the purpose of performingthis invention. Preferably, the amplification vector has additionallyother sequences encoding viral functions for host infectivity,cell-to-cell and/or systemic movement for spreading throughout the plantand for further increasing the frequency of targeted transformation. Theamplification vector may have further sequences for functions such asintegration into the host chromosome, viral particle assembly, controlof gene silencing by the host, and/or control of host physiology.Alternatively, such additional viral functions may be provided on anadditional vector. The additional vector may be a replicating vector aswell. Preferably, the additional vector is a non-replicating vector suchthat the additional viral functions are only transiently expressed. Thismay reduce disease symptoms of the plant. Further, the amplificationvector may be of retrotransposon origin.

The amplification vector may further contain a DNA sequence of intereste.g. a gene to be expressed e.g. for conferring a useful trait, forperforming mutagenesis etc.

Said site-specific or homologous recombination takes place between theamplification vector and a host nuclear DNA. Said host nuclear DNA maybelong to a nuclear chromosome of the host or it may belong to anepisomal nuclear DNA. Preferably, said recombination takes place betweenthe amplification vector and a DNA on a nuclear chromosome of the host.

In order to facilitate site-specific or homologous recombination,suitable recombination enzymes such as site-specific recombinases,restriction enzymes or integrases may be provided from an additionalvector or from a gene previously incorporated into said plant. Such anadditional vector may be co-transformed with the amplification vector orit may be transformed separately. Expression of the recombination enzymemay be constitutive or inducible. Preferably, the recombination enzymeis only transiently expressed e.g. from a non-replicating vector. If therecombination enzyme is present at the target locus of the nucleargenome, its function may be destroyed as a result of the recombinationevent.

In case of homologous recombination, a recombination enzyme may not haveto be provided externally and the process may rely on an endogenousrecombinase. However, the efficiency may be further increased byadditionally providing a recombination enzyme for promoting homologousrecombination. Such an enzyme may be an enzyme native to said plant, aheterologous enzyme or an engineered enzyme.

If homologous recombination is used to target a DNA of interest into thenuclear genome of the plant, any site in the nuclear genome may betargeted as long as suitable selection means exist to select for thedesired recombination event. Selection may be achieved by introducing amutation conferring an antibiotic or inhibitor resistance or byproviding a resistance marker gene. As more genome sequences becomeknown, targeting of a desired site by homologous recombination becomesmore broadly applicable.

A preferred embodiment of targeted homologous recombination issite-directed mutagenesis of a gene of the plant nuclear genome. Forthis purpose, the amplification vector may contain the desired mutationflanked by homologous sequences of the target site.

If site-specific recombination is used to target a DNA of interest intothe nuclear genome of the plant, target site(s) recognizable bysite-specific recominases are preferably pre-introduced into the plantaccording to the above five-step process. The above five-step processcomprises two stages: in the first stage (step (i) and (ii)), atransgenic plant having pre-engineered target sites for site-specificrecombination is produced. Preferably, the target sites are stablyincorporated into the nuclear genome. Transfecting or transforming saidfirst DNA in step (i) of the five-step process may be non-targeted. Manytransgenic plants with target sites introduced in many different loci ofthe genome may be produced. Then a transgenic plant line with the targetsite at a desired location may be chosen for performing the steps of thesecond stage (steps (iii) to (v)). Integration of a DNA of interest inthe second stage can then be targeted. According to this process, stabletransgenic plant lines may be produced in the first phase. Each suchtransgenic plant line may then be used for various purposes according tothe second stage, making this process highly versatile. At least one ofsaid first or said second DNA is delivered by an amplification vector.Preferably, at least said second DNA is delivered by an amplificationvector.

Both said first and said second DNA may comprise a sequence of interest.Such a sequence of interest may be a selectable marker and/or a gene tobe expressed e.g. for conferring the plant with a useful trait.Preferably, the recombination event may establish a functional sequence.An example for the establishment of a functional sequence is theplacement of a DNA to be expressed under the control of a promoter,whereby the promoter may be provided by said first or said second DNAand the DNA to be expressed may be provided by said second or said firstDNA, respectively. Further, other functions necessary for functionalexpression of a gene such as combination of two fragments of a codingsequence may be combined by said recombination event. The recombinationevent may also be used to destroy the function of a gene or to eliminatea sequence at the target site.

Said plant cells may be provided with said amplification vector (e.g. areplicon) or with (a) precursor(s) thereof (a pre-replicon orpro-vector). If said plant cells are provided with said precursor, theprecursor has to be adopted to be processed to said amplification vectorin the plant cell. The amplification vector may e.g. be excised from aprecursor by recombination. However, if an amplification vector is to beexcised from a precursor, this is preferably achieved by providing theprecursor with two origins of replication for allowing replicativerelease of the amplification vector. Excision of the amplificationvector from a precursor is preferably done in combination withAgrobacterium transformation for excising the amplification vector outof the Ti-plasmid delivered by Agrobacterium. Further, the amplificationvector may be assembled in plant cells from two or more precursors byrecombination.

Said plant cells may be provided with said amplification vector or itsprecursor by several methods. Preferred methods areAgrobacterium-mediated delivery, direct viral transfection, andnon-biological delivery (e.g. particle bombardment). In direct viraltransfection, infectious viral material is directly applied to planttissue. Direct viral transfection should be distinguished fromAgroinfection where viral DNA is delivered indirectly usingAgrobacterium. In Agrobacterium-mediated delivery, Ti-plasmids aredelivered as precursors of amplification vectors, which are processed inthe plant cell to generate said amplification vectors. Direct viraltransfection and non-biological delivery methods are preferred.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A-F) shows six of many possible ways to increase the frequency ofsite-targeted or homologous recombination events in plant cells.

X—donor molecule or sequence of interest; Y—acceptor or target site;Z—frequency of site-targeted or homologous recombination events;W—frequency of non-homologous recombination or random integrationevents. Larger letters mean increased number/concentration of molecules(X, Enzymes), target sites (Y) and increased frequency of recombinationevents (Z, W).

FIG. 2 depicts the scheme of experiment designed to test the ability ofa geminivirus-based vector to replicate.

FIG. 3 depicts the scheme for comparing the efficiencies ofsite-specific recombination events using replicating and non-replicatingvectors in transient expression experiments.

FIG. 4 depicts the scheme for comparing the efficiencies ofsite-directed recombination events in transgenic plant cells usingreplicating and non-replicating vectors with donor sequences of interest(GFP). Site-specific Cre recombinase is provided transiently from anon-replicating vector.

FIG. 5 depicts the scheme for comparing the efficiencies ofsite-directed recombination events in transgenic plant cells usingreplicating and non-replicating vectors with donor sequences of interest(BAR). Site-specific Cre recombinase is provided transiently fromnon-replicating vector.

FIG. 6 depicts the scheme for comparing the efficiencies ofsite-directed recombination events in transgenic plant cells usingreplicating and non-replicating vectors with donor sequences of interest(BAR). Site-specific Cre recombinase is provided transiently togetherwith replicase from a non-replicating vector.

FIG. 7 depicts the scheme for comparing the efficiencies ofsite-directed recombination events in transgenic plant cells usingreplicating vectors with ability for cell-to-cell movement (due to thereplication and movement of BGMV B genome) and non-replicating vectorswith donor sequences of interest (GFP). Site-specific Cre recombinase isprovided transiently from replicating or non-replicating vectors.

FIG. 8 depicts the scheme for comparing the efficiencies ofsite-directed recombination events in transgenic plant cells usingreplicating vectors with the ability for cell-to-cell movement (but BGMVB genome is unable to move) and non-replicating vectors with donorsequences of interest (GFP). Site-specific Cre recombinase is providedtransiently from replicating or non-replicating vectors.

FIG. 9 depicts the scheme for comparing the efficiencies ofsite-directed recombination events in transgenic plant cells usingreplicating vectors that retain the ability for cell to cell movement(due to the replication and movement of BGMV B genome andnon-replicating vectors with donor sequences of interest (GFP).Site-specific Cre recombinase is expressed by transgenic plant cells andis switched off as a result of site-directed recombination.

FIG. 10 depicts the scheme of experiments for site-directed mutagenesisby homolgous recombination using geminivirus-based replicating vector.

FIG. 11 depicts the T-DNA based constructs pICH5203, pICH4300, pICH4699,and pICH5170 made to demonstrate amplification of replicons with thereplicase provided in trans and shows results of a Southern blotanalysis.

FIG. 12 depicts T-DNA based constructs pICH6272, pICH6313, pICH7555,pICH5170 and pICH6040 designed for site specific integration using thephage C31 integrase system.

FIG. 13 depicts constructs pICH4371, pICH4461, pICH7311, pICH5170, andpICH1754 used to demonstrate increased site-specific recombinationbetween 5′ and 3′ provectors using geminivirus-mediated 3′ end provectoramplification. Also, a picture of an infiltrated N. benthamiana leafshowing increased site-specific recombination is shown.

FIG. 14 depicts the T-DNA based constructs pICH7477, pICH7480, pICH7499,pICH5170, and pICH7500 designed for homologous recombination usingpro-vector elements as the detection system of recombination events.

Appendices 1 to 16 depict vectors used in the examples section.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the use of amplification vectors toincrease the efficiency of targeted transformation in plants. Vectorscapable of replication in a plant cell that amplify passenger DNA (DNAof interest) in cells into which the DNA has been delivered, are shownto greatly enhance the frequency of directed recombination. In addition,when the vectors used are derived from viral genomes and retain otherviral capabilities such as cell-to-cell or long distance (systemic)movement, the passenger DNA to be targeted can be transported toadjacent cells and throughout the organism where it also replicates; theresulting targeted recombination effect amplifies even further. We havefound that increased homologous recombination frequencies are obtainedwith replicating vectors at either natural or pre-engineered targetsites using either the endogenous recombination machinery of the plantor heterologous site-specific recombination systems.

Irrespective of whether the incoming DNA needs to be recombined usingthe endogenous recombination machinery or heterologous site-specificrecombinases, recombination theoretically involves a physicalinteraction between incoming DNA molecules and the target site.Therefore, it will be dependent on the relative concentrations ofincoming and target DNA (Wilson et al., 1994, Proc. Natl. Acad. Sci.,91, 177-181). This is particularly important when recombinases such asCre are used, since the recombination reaction (which is bimolecular)takes place at a much lower rate than the excision reaction andsophisticated strategies (described above) have to be used to recover aninsertion event. Different approaches already have been or can beundertaken (see FIG. 1). Our approach to modify the efficiency ofsite-specific recombination consists of using replicating vectors toamplify the DNA to be targeted with or without the increase of theconcentration of an enzyme involved in homologous recombination.Optionally, expression of proteins involved in non-homologousrecombination may additionally be suppressed. Amplification vectors areshown herein to replicate passenger DNA within the cells into which theyare delivered. If virus-based amplicons (replicons) are used, theinfection will spread to adjacent cells further improving the efficiencyof targeted insertions. The unexpected outcome is an enormouslyincreased targeted transformation frequency. The success of thisapproach was surprising, since a competing repair mechanism callednon-homologous end-joining (NHEJ) also takes place at a high frequencyin most higher plant species including all economically important crops.NHEJ is one of the reasons why selection for the desired site-targetedevents diluted or hidden in a strong background of unwanted reactions isdifficult in the prior art.

Targeted transformation according to this invention makes plantengineering a much more precise, controlled and efficient technology. Itis broadly applicable and it allows to solve many current problems inplant genetic engineering including gene introduction duration, lack ofcontrol over gene activation, gene silencing, vector design limitations,single-step nature of current engineering processes, line conversionduration and associated linkage drag, etc. To our knowledge, there is noprior art for the use of amplification vectors for targetedtransformation in plants.

Vectors Utilizing Plant Viral Amplicons

Geminiviruses are members of a large and diverse family ofplant-infecting viruses characterized by twinned icosahedral capsids andcircular, single-stranded DNA genomes (For reviews, see Timmermans etal., 1994, Annu. Rev. Plant Physiol. Plant Mol. Biol., 45, 79-113;Mullineaux et al., 1992, in: Wilson, T. M. A., Davies, J. W. (Eds.)Genetic Engineering with Viruses, CRC Press, Boca Raton, Fla., 187-215;Palmer & Rybicki, 1997, Plant Science, 129, 115-130). Geminiviruses canbe generally classified into two subgroups (with the exception of a fewatypical geminiviruses):

-   (i) monopartite geminiviruses which have a single component genome,    infect monocotyledonous plants and are transmitted by leafhoppers,    and-   (ii) bipartite geminiviruses whose genome is composed of two    circular genomes, infect dicotyledonous plants and are transmitted    by whiteflies.    Some examples of monopartite geminiviruses include the maize streak    virus (MSV), the wheat dwarf virus (WDV), the Digitaria streak virus    (DSV), and the Miscanthus streak virus (MiSV). Examples of bipartite    geminiviruses include the tomato golden mosaic virus (TGMV), the    bean golden mosaic virus (BGMV), the African cassaya mosaic virus    (ACMV), and the abutilon mosaic virus (AbMV).

Geminiviruses replicate their genomes using a rolling-circle mechanismsimilar to that used by ssDNA containing coli phages (e.g. PhiX174)(Saunders et al., 1991, Nucl. Acids. Res., 19, 2325-2330; Stenger etal., 1991, Proc. Natl. Acad. Sci., 88, 8029-8033). A consequence of thismode of replication is the generation of double-stranded DNA genomes asreplication intermediates. These double-stranded DNA genomes behaveessentially as high copy plant plasmids and can be present at extremelyhigh copy numbers of up to 30000 copies per nucleus of infected cell(Kanevski et al., 1992, Plant J., 2, 457-463; Timmermans et al., 1992,Nucl. Acids Res., 20, 4047-4054). These characteristics and the factthat, collectively, geminiviruses have a very broad host range, hasstimulated a lot of research in developing geminiviruses as replicatingvectors for plants, mainly to enhance levels of transgene expression orto develop resistance strategies against geminiviral diseases. Severalpatents have been issued which describe the use of replicatinggeminivirus vectors for enhancing gene expression in plants (U.S. Pat.No. 5,981,236, WO020557A2, U.S. Pat. No. 6,110,466, U.S. Pat. No.6,147,278, U.S. Pat. No. 6,077,992), for developing plant diseaseresistance strategies (some examples are U.S. Pat. No. 6,118,048,WO9739110A1, U.S. Pat. No. 6,133,505, U.S. Pat. No. 6,087,162), or forsuppressing gene expression in plants (WO9950429A1).

There are several publications that describe attempts of combined use ofgeminivirus vectors and transposons to achieve transposition andtransformation of genomes of monocots (Laufs et al., 1990, Proc. Natl.Acad. Sci. USA., 87, 7752-7756; Shen & Hohn, 1992, Plant J., 2, 35-42;Sugimoto et al., 1994, Plant J., 5, 863-871). One publication reportsthe use of geminiviruses as amplification vectors to increasetransformation frequency (Sugimoto et al., 1994, Plant J., 5, 863-871).This works is inspired by a Drosophila transformation method which isbased on transposition of P elements from introduced DNA molecules tochromosomal DNA. The authors cloned a Ds element and the Ac transposasein separate geminivirus miscanthus streak virus (MiSV) vectors andco-bombarded rice protoplasts with these vectors. After excision, a lowfrequency of reinsertion (in the order of 10⁻⁵) led to the recovery offive chromosomal insertion events. No transposition event could bedetected in a control non-replicating vector, indicating thatreplication was required to recover reinsertion events due to the lowtransposition frequency. This approach differs from our invention by thenon-targeted nature of the resulting transformation events.

The present invention preferably uses replicons as amplification vectors(replicons are freely replicating circular DNA molecules, the use ofwhich is described in many publications, see reviews: Timmermans et al.,1994, Annu. Rev. Plant. Physiol. Plant Mol. Biol., 45, 79-113;Mullineaux et al., 1992, in: Wilson, T. M. A., Davies, J. W (Eds.)Genetic Engineering with Viruses, CRC Press, Boca Raton, Fla., 187-215;Palmer & Rybicki, 1997, Plant Science, 129, 115-130). Replicons containa geminivirus origin of replication and preferably a DNA sequence ofinterest. Replication is mediated by the geminiviral replicase which canbe present either on the replicon itself, on a co-transformedreplicating or non-replicating plasmid, or it may be expressed from astably transformed expression cassette integrated into a chromosome.Replicons may be cloned in bacteria in the form of pre-replicons.Replicons may be released from pre-replicons by either one of twoapproaches: (i) by digesting the pre-replicon with an enzyme that willrelease replicon DNA from a plasmid vector or (ii) by usingpre-replicons containing more than one unit length of genome. In thefirst approach, excised DNA will recircularize after its introductioninto cells using an endogenous ligase (Bisaro et al., 1983, Nucl. Acids.Res., 11, 7387-96). In the second approach, circular replicons arereleased from pre-replicons by homologous intramolecular recombinationin duplicated sequences or by a replicational release mechanism(provided that two origins of replication are present in thepre-replicon) (Stenger et al., 1991, Proc. Natl. Acad. Sci., 88,8029-8033; Rogers et al., 1986, Cell, 45, 593-600). A pre-repliconcontains a replicon in its continuity and replicon formation is theprocess of release of said continuity from flanking sequences of saidpre-replicon. Replicon(s) can also be formed in a plant host fromprecursor vector(s) or pro-vector(s). Precursor vector(s) orpro-vector(s) are nucleic acids, which upon processing in plant hostform vector(s) that are able to amplify and express heterologous nucleicacid sequence(s) in said host. Said processing includes formation ofcontinuity from discontinued vector parts.

Replicons can be introduced into plant cells by a variety of mechanismsincluding Agrobacterium-mediated transformation, electroporation,particle delivery or any other DNA delivery technology. Alternatively,the replicon can be released from a pre-replicon integrated in achromosome. Pre-replicons in these constructs will contain two originsof replication so as to facilitate release of replicons by a replicativerelease mechanism. Release of the replicon and replication will becontrolled by expression of the replicase. It will therefore be usefulto be able to control the timing of expression by using an inducible ortissue-specific promoter in order to minimize the potential detrimentaleffect of replicon replication on cell survival.

Although geminivirus-based amplification vectors are preferred forperforming this invention, other vectors capable of amplification inplant cells may also be used for this invention.

Both RNA- and DNA-containing viruses could be used for the constructionof replicating vectors, and examples of different viruses are given inthe following list:

DNA Viruses:

Circular dsDNA Viruses: Family: Caulimoviridae, Genus: Badnavirus, Typespecies: commelina yellow mottle virus, Genus: Caulimovirus Typespecies: cauliflower mosaic virus, Genus “SbCMV-like viruses”, Typespecies: Soybean chloroticmottle virus, Genus “CsVMV-like viruses”, Typespecies: Cassaya vein mosaicvirus, Genus “RTBV-like viruses”, Typespecies: Rice tungro bacilliformvirus, Genus: “Petunia veinclearing-like viruses”, Type species: Petunia vein clearing virus;Circular ssDNA Viruses: Family: Geminiviridae, Genus: Mastrevirus(Subgroup I Geminivirus), Type species: maize streak virus, Genus:Curtovirus (Subgroup II Geminivirus), Type species: beet curly topvirus, Genus: Begomovirus (Subgroup III Geminivirus), Type species: beangolden mosaic virus;RNA Viruses:ssRNA Viruses: Family: Bromoviridae, Genus: Alfamovirus, Type species:alfalfa mosaic virus, Genus: Ilarvirus, Type species: tobacco streakvirus, Genus: Bromovirus, Type species: brome mosaic virus, Genus:Cucumovirus, Type species: cucumber mosaic virus;Family: Closteroviridae, Genus: Closterovirus, Type species: beetyellows virus, Genus: Crinivirus, Type species: Lettuce infectiousyellows virus, Family: Comoviridae, Genus: Comovirus, Type species:cowpea mosaic virus, Genus: Fabavirus, Type species: broad bean wiltvirus 1, Genus: Nepovirus, Type species: tobacco ringspot virus;Family: Potyviridae, Genus: Potyvirus, Type species: potato virus Y,Genus: Rymovirus, Type species: ryegrass mosaic virus, Genus: Bymovirus,Type species: barley yellow mosaic virus;Family: Sequiviridae, Genus: Sequivirus, Type species: parsnip yellowfleck virus, Genus: Waikavirus, Type species: rice tungro sphericalvirus; Family: Tombusviridae, Genus: Carmovirus, Type species: carnationmottle virus, Genus: Dianthovirus, Type species: carnation ringspotvirus, Genus: Machlomovirus, Type species: maize chlorotic mottle virus,Genus: Necrovirus, Type species: tobacco necrosis virus, Genus:Tombusvirus, Type species: tomato bushy stunt virus, Unassigned Generaof ssRNA viruses, Genus: Capillovirus, Type species: apple stem groovingvirus;Genus: Carlavirus, Type species: carnation latent virus; Genus:Enamovirus, Type species: pea enation mosaic virus,Genus: Furovirus, Type species: soil-borne wheat mosaic virus, Genus:Hordeivirus, Type species: barley stripe mosaic virus, Genus:Idaeovirus, Type species: raspberry bushy dwarf virus;Genus: Luteovirus, Type species: barley yellow dwarf virus; Genus:Marafivirus, Type species: maize rayado fino virus; Genus: Potexvirus,Type species: potato virus X;Genus: Sobemovirus, Type species: Southern bean mosaic virus, Genus:Tenuivirus, Type species: rice stripe virus,Genus: Tobamovirus, Type species: tobacco mosaic virus,Genus: Tobravirus, Type species: tobacco rattle virus,Genus: Trichovirus, Type species: apple chlorotic leaf spot virus;Genus: Tymovirus, Type species: turnip yellow mosaic virus; Genus:Umbravirus, Type species: carrot mottle virus; Negative ssRNA Viruses:Order: Mononegavirales, Family: Rhabdoviridae, Genus: Cytorhabdovirus,Type Species: lettuce necrotic yellows virus, Genus: Nucleorhabdovirus,Type species: potato yellow dwarf virus;Negative ssRNA Viruses: Family: Bunyaviridae, Genus: Tospovirus, Typespecies: tomato spotted wilt virus;dsRNA Viruses: Family: Partitiviridae, Genus: Alphacryptovirus, Typespecies: white clover cryptic virus 1, Genus: Betacryptovirus, Typespecies: white clover cryptic virus 2, Family: Reoviridae, Genus:Fijivirus, Type species: Fiji disease virus, Genus: Phytoreovirus, Typespecies: wound tumor virus, Genus: Oryzavirus, Type species: rice raggedstunt virus;Unassigned Viruses: Genome ssDNA: Species: banana bunchy top virus,Species coconut foliar decay virus, Species: subterranean clover stuntvirus,Genome: dsDNA, Species: cucumber vein yellowing virus; Genome: dsRNA,Species: tobacco stunt virus,Genome: ssRNA, Species Garlic viruses A,B,C,D, Species grapevine fleckvirus, Species maize white line mosaic virus, Species olive latent virus2, Species: ourmia melon virus, Species Pelargonium zonate spot virus;Satellites and Viroids: Satellites: ssRNA Satellite Viruses: Subgroup 2Satellite Viruses, Type species: tobacco necrosis satellite,Satellite RNA, Subgroup 2 B Type mRNA Satellites, Subgroup 3C Typelinear RNA Satellites, Subgroup 4 D Type circular RNA Satellites,Viroids, Type species: potato spindle tuber viroid.

Mostly, vectors of plant viral origin are used as plasmids capable ofautonomous replication in plants, but the principles necessary forengineering such plasmids using non-viral elements are known. Forexample, many putative origins of replication from plant cells have beendescribed (Berlani et al., 1988, Plant Mol. Biol., 11, 161-162;Hernandes et al., 1988, Plant Mol. Biol., 10, 413-422; Berlani et al.,1988, Plant Mol. Biol, 11, 173-182; Eckdahl et al., 1989, Plant Mol.Biol., 12, 507-516). It has been shown that the autonomously replicatingsequences (ARS elements) from genomes of higher plants have structuraland sequence features in common with ARS elements from yeast and higheranimals (Eckdahl et al., 1989, Plant Mol. Biol, 12, 507-516). The plantARS elements are capable of conferring autonomous replicating ability toplasmids in Saccharomyces cerevisiae. Studies of maize nuclear DNAsequences capable of promoting autonomous replication of plasmids inyeast showed that they represent two families of highly repeatedsequences within the maize genome. Those sequences have characteristicgenomic hybridization pattern. Typically there was only one copy of anARS-homologous sequence on each 12-15 kb of genomic fragment (Berlani etal., 1988, Plant Mol. Biol., 11:161-162). Another source of replicons ofplant origin are plant ribosomal DNA spacer elements that can stimulatethe amplification and expression of heterologous genes in plants(Borisjuk et al., 2000, Nature Biotech., 18, 1303-1306).

Therefore, an amplification vector contemplated in this invention is notnecessarily derived from a plant virus. Similarly, plant DNA virusesprovide an easy way of engineering amplification vectors that could beespecially useful for targeted DNA transformation, but vectors madeentirely or partially of elements from plant RNA viruses or evennon-plant viruses are possible. Advantages of plant-virus based vectorsare evident. Such vectors, in addition to amplification, may provideadditional useful functions such as cell-to-cell and long distancemovement. Further, they can frequently more easily removed from theplant cell aposteriori by using known methods of virus eradication frominfected plants.

In the present invention, replicons are preferably used to increase thecopy number of a desired target sequence in the nuclei of the hostcells. In one embodiment of this invention, recombination with a targetsite will occur by the intermediate of specific recombination sitesplaced on the replicon and at the target site. In another embodiment,recombination will occur as a result of homologous recombination betweensequences carried by the replicon and homologous sequences in the hostgenome. Details of the vectors and uses of these vectors are describednext.

Replicons Containing Recombination Sites from Heterologous RecombinationSystems

Suitable recombinases/recombination site systems include inter alia theCre-Lox system from bacteriophage P1 (Austin et al., 1981, Cell, 25,729-736), the Flp-Frt system from Saccharomyces cerevisiae (Broach etal., 1982, Cell, 29, 227-234), the R-Rs system from Zygosaccharomycesrouxii (Araki et al., 1985, J. Mol. Biol., 182, 191-203), the integrasefrom the Streptomyces phage PhiC31 (Thorpe & Smith, 1998, Proc. Natl.Acad. Sci., 95, 5505-5510; Groth et al., 2000, Proc. Natl. Acad. Sci.,97, 5995-6000), and resolvases. One or two recombination sites may bepresent on the replicon. When a single site is present, recombinationwill lead to integration of the entire replicon at the target siteincluding geminiviral sequences (one-sided recombination). Preferably,two recombination sites flanking the DNA to be targeted are thereforeemployed (two-sided recombination). Upon expression of the recombinase,recombination of the two sites with compatible sites at the target locuswill lead to the replacement of the DNA sequence located between therecombination sites at the target locus by the DNA sequence of intereston the replicon. Selection for targeted events can easily beaccomplished e.g. by including a promoterless selection marker on theDNA fragment to be targeted and a promoter at the target site.Recombination will then result in activation of the selectable markergene by placing it under the control of the promoter at the target site,thus establishing a functional marker. The opposite strategy wherein apromoterless selectable marker is present at the target site and apromoter on the replicon is also possible.

When two recombination sites are present on the replicon, it isadvantageous that these sites do not recombine with each other sincethis may delete the sequence of interest during replication of thereplicon. Pairs of recombination sites that cannot recombine with eachother have been described for the Cre-Lox and Flp/Frt systems. Suchsites, called heterospecific sites, contain mutations in the centralcore region. These sites can recombine at wild-type level with sitesidentical to them but not with different heterospecific sites (Bethke &Sauer, 1997, Nucl. Acids Res., 25, 2828-2834, see also example 2).Recombination sites of some systems, such as the PhiC31 integrase cannotrecombine with identical sites, but only with different compatiblesites. For example, in the presence of the PhiC31 integrase, attP sitesrecombine with an attB sites, thus producing attL and attR. attP or attBsites may be used on the replicon, while compatible sites may be placedat the target sites on the genome.

Target sites in the plant nuclear genome may be naturally occurring(resident genes to be targeted, sequences recognized by heterologoussite-specific recombinases, restriction enzymes, etc.) or pre-engineeredand introduced into the plant genome using existing technologies.Various methods may be used for the delivery of such sites into plantcells such as direct introduction of a vector into the plant cell bymeans of microprojectile bombardment (U.S. Pat. No. 05,100,792; EP00444882B1; EP 00434616B1), electroporation (EP00564595B1; EP00290395B1;WO 08706614A1) or PEG-mediated treatment of protoplasts. These threemethods may be summarized as non-biological delivery methods.Agrobacterium-mediated plant transformation (U.S. Pat. No. 5,591,616;U.S. Pat. No. 4,940,838; U.S. Pat. No. 5,464,763) also presents anefficient way of vector delivery. In principle, other planttransformation methods may also be used such as microinjection (WO09209696; WO 09400583A1; EP 175966B1). The choice of the transformationmethod depends on the kind of plant species to be transformed. Forexample, for monocot transformation, the microprojectile bombardment ispreferable, while for dicots, Agrobacterium-mediated transformationgives better results in general. The same methods may be used fortransfecting or transforming a plant cell with an amplification vectoror for said providing a plant cell with DNA. Moreover, this may beachieved by viral transfection or by using a vector or pro-vector thatwas pre-integrated into the plant nuclear DNA to form an autonomouslyreplicating plasmid.

An appropriate heterologous recombinase may be expressed either from thereplicon, from a co-transformed replicating or non-replication plasmid,or it may be expressed from the chromosomal target site. Its expressioncan be made constitutive, tissue-specific or inducible. Variouspossibilities are illustrated in the examples section below.

Bipartite geminiviruses have two genome components, DNAA and DNAB. The Bgenome encodes two genes whose products are required for cell-to-celland systemic movement of both genome components (Brough et al., 1988, J.Gen. Virol., 69, 503-514; Qin et al., J. Virol., 72, 9247-9256). Anexample is the DNAB genome of BGMV, which encodes two open readingframes, BL1 and BR1. Expression of genes encoded on the B genome willallow replicons to move from cell to cell or systemically. Both genesmay be provided by co-transforming a construct from which a wild-type Bgenome will be released. Alternatively, B genes can be provided on anon-replicating plasmid. In this way, genes of the B genome may beexpressed transiently until the non-replicating plasmid disappears fromthe cell. This is advantageous as expression of the genes of the Bgenome and in particular BL1 is responsible for the disease symptoms ofgeminivirus-infected plants (Pascal et al., 1993, Plant Cell, 5,795-807). It has also been shown that transient expression of genesencoded by the B genome is sufficient for systemic movement of the DNAAgenome for TGMV (Jeffrey et al., 1996, Virology, 223, 208-218).

Replicons Carrying Sequences with Homology to Endogenous Sequences

Replication of replicons containing DNA sequence(s) which are homologousto endogenous sequences will increase recombination with homologoustarget sequences. Homologous recombination is preferably initiated bydouble strand breaks or nicks in DNA. Geminiviral DNA is present incells in different forms including supercoiled double-stranded circular,open-circular, and linear DNA (Saunder et al., 1991, Nucl. Acids Res.,19, 2325-2330). Nicks in open-circular DNA and double strand breaks onlinear DNA will induce homologous recombination events. To furtherincrease recombination, it is also possible to induce the formation ofdouble strand breaks in replicated DNA by placing on the replicon one ortwo restriction sites for a rare cutting enzyme such as the yeast HO orI Sce-I endonucleases. The endonuclease can be expressed from aco-transformed replicating or non-replicating plasmid or from a stablyintegrated expression cassette integrated in a chromosome. Itsexpression can be constitutive, tissue-specific or inducible.

The vector used in this invention may be a pro-vector. A pro-vector is avector from which a vector according to the invention is generatedwithin a plant cell by the plant nucleid acid processing machinery, e.g.by intron splicing.

EXAMPLES

The following examples demonstrate, inter alia, the detection ofsite-targeted integration events at increased frequency due toreplicating amplification vectors. Further, examples for successfulselection of progeny cells and recovery of transformants preferablyusing transiently replicating amplification vectors are given.

Example 1

This example reports the cloning of replicating clones of BGMV DNAA andDNAB genomes (FIG. 2).

Cloning of a DNAA Genome Replicating Vector Containing GFP:

pUC19 DNA was amplified with primers dnaapr7 (aac tgc agt cta gac tggccg tcg ttt tac aac) and dnaapr8 (aac tgc aga aca att gct cga ggc gtaatc atg gtc a), and the amplified fragment digested with Pst1 andreligated. The resulting plasmid, pIC1144, is similar to pUC19, but thepolylinker has been replaced with Xho1, MfeI, and Pst1. DNA wasextracted from Phaseolus vulgaris tissue infected by bean golden mosaicvirus (BGMV) isolate DSMZ PV-0094 obtained from the German Collection ofMicroorganisms and Cell Cultures (DSMZ, Deutsche Sammlung vonMikroorganismen und Zelikulturen GmbH). A fragment of the genomeencompassing the BGMV common region. (CR; contains the BGMV origin ofreplication) was amplified by PCR with primers dnaapr3 (ggg aat tca ctagta aag atc tgc cgt cga ctt gga att g) and dnaapr4 (caa tgc atc atg gcgcat cac gct tag g) and cloned as an EcoRI-NsiI fragment in pIC1144digested with MfeI and PstI, resulting in plasmid pIC1156. The BGMVinsert in pIC1156 was sequenced. Two other BGMV DNAA genome fragmentswere amplified from BGMV infected Phaseolus vulgaris DNA with primerspairs dnaapr9 (aag ctg cag aag gat cct ctg gac tta cac gtg gaatgg)/dnaapr13 (cgc tcg agg ccg tcg act tgg aat tgt c), and dnaapr5 (gaagat ctg caa gag gag gtc agc a)/dnaapr10 (aag ctg cag atc tat ttc tat gattcg ata acc). The sum of these fragments amounts to a complete BGMVgenome without the coat protein. These fragments were digested withXho1/Pst1 and Pst1/BgIII (respectively) and cloned in a threeway-ligation in pIC1156 digested with XhoI and BgIII. The resultingplasmid contains one complete BGMV DNAA genome without the coat proteingene flanked by duplicated BGMV DNAA common regions. Three clones werekept for testing: pIC1663, 1664 and 1667. A multicloning site containingBamHI and PstI replaces the coat protein gene.

A GFP (SGFP stands for synthetic GFP) coding sequence was cloned as aBamHI-PstI fragment from pIC011 (Hbt promoter-Synthetic GFP codingsequence-Nos terminator in pUC18), into the BamHI-Pst1 sites of pIC1663pIC1664 and pIC1667, resulting in plasmids pIC1693, pIC1694 and pIC1697(Appendix 1). GFP is placed under the control of the coat proteinpromoter.

A DNAA genome clone mutated for the replicase was made by destroying aBgIII site present in the AL1 ORF. As two BgIII sites are present inpIC1693, pIC1694 or pIC1697, an intermediate construct lacking thesecond BgIII site was made (pIC2690). This construct was made byamplifying a fragment from pIC1694 by PCR using primers dnaapr16 (aagctg cag gtc tat ttc tat gat tcg ata acc) and dnaapr5 (gaa gat ctg caagag gag gtc agc a), and cloning a Pst1-HindIII fragment from theamplified product into pIC1694 digested with Hind3 and PstI. pIC2690 wasthen digested with BgI2, the ends filled-in with klenow polymerase andreligated to give plasmid pIC2705 (Appendix 3).

Cloning of the DNAB Genome

A complete DNAB genome was amplified by PCR from BGMV-infected Phaseolusvulgaris DNA with primers dnabpr2 (cgg cat gca tgc att tgg agg att tgctaa ctg) and dnabpr3 (cgg atg cat tca att atg tag agt cac aca g). Theamplified fragment was cloned in the pGEMT vector from promega.Digestion of the clones with NsiI releases a complete linear DNABgenome. Twelve colonies were picked and nine clones containing aninsert, pIC1911 to pIC1919 (Appendix 2), were kept for testing forfunctionality.

Test for Functionality of DNAA and DNAB Clones:

To test for functionality of GFP (functional coat protein promoter andfunctional coding sequence), pIC1693, pIC1694 and pIC1697 were bombardedin Nicotiana benthamiana and Phaseolus vulgaris excised leaves using aBiolistic Particle Delivery System 1000/HE (Biorad). GFP-expressingepidermal cells could be detected the next day in leaves of both speciesfor all three constructs.

To test for replication and movement of DNAA and DNAB clones, pIC1693,1694 and 1697 were cobombarded with pIC1911 to 1919 (digested with NsiI)in pairwise combinations, in Phaseolus vulgaris excised leaves. Allcombinations gave rise to hundreds of GFP expressing cells. For twoplasmid combinations, 1694/1914 and 1697/1919, expression of GFP spreadto neighbouring cells for a few of the GFP expressing cells, mainly inveins.

To test for the functionality of the DNAA and DNAB clones in entireplants, combinations of pIC1694/1914 and pIC1697/1919 were bombarded inthe radicle of germinating bean plants (FIG. 2). The seedlings weretransferred to soil and scored for GFP expression in the first twoleaves 10 days later. The majority of the seedlings showed fluorescencein some of the veins of the first two leaves. DNA was extracted from thefirst two leaves and analyzed by Southern blotting with a DNAA probe.Single stranded, supercoiled double stranded and open circle doublestranded forms were detected when plants were inoculated with the DNAAand DNAB clones but not when the plants were inoculated with DNAA clonesonly. The GFP protein was also detected by Western blotting using a GFPantibody.

Example 2

This example shows that replication of a plasmid can increase thefrequency of recombination with a target co-transformed non-replicatingplasmid. In this example, recombination is mediated by Cre recombinaseand takes place at the loxP and LoxM sites present on both the donor andrecipient plasmid (FIG. 3).

Description of the Plasmids:

A PCR fragment was amplified from pIC1667 with dnaapr13 (cgc tcg agg ccgtcg act tgg aat tgt c) and dnaapr15 (ccc atg cat cta gag tta acg gcc ggccca aat atc taa cgt tct cac atg) and cloned as an XhoI-NsiI fragment inpIC1667 digested with XhoI and PstI. The resulting plasmid, pIC1951, issimilar to pIC1667 but lacks the coat protein gene promoter.

Plasmid pIC551 was obtained by (i) performing PCR on pUC119 digestedwith XbaI and Hind3 with primers adlox1 (gtt cta gat gtt aac ggc gcg ccggcg taa tca tgg tca), adlox2 (aac cat gga gaa ttc ggc cgg ccc tgg cogtcg ttt tac aac), adlox3 (cgg gat cct gag ctc tat aac ttc gta taa tgtatg cta tac gaa gtt gtt cta gat gtt aac gg) and adlox4 (cgg gat ccc tgcaga taa ctt cgt ata atc tat act ata cga agt tag aaa aac aac cat gga gaattc gg), (ii) digesting the PCR product with BamHI and (iii) religatingthe digested fragment. pIC551 is similar to pUC119 but has thepolylinker AscI-HpaI-XbaI-loxA-SacI-BamHI-PstI-LoxM-NcoI-EcoRI-FseI.LoxA(acaacttcgtatagcatacattatacgaagttat) and LoxM (ataacttcgtataatctatactatacgaagttag) are modified LoxP sites. LoxA differs from LoxP at onenucleotide in one of the inverted repeats and can recombine at wild-typelevel with LoxP. LoxM has two mutations in the central spacer region andcannot recombine with either LoxP or LoxA, but can freely recombine withitself as is the case for other heterospecific sites (Bethke & Sauer,1997, Nucl. Acids Res., 25, 2828-2834).

A BamHI-Pst1 fragment from pIC011 containing the GFP coding sequence wascloned in the BamHI and Pst1 sites of pIC551, resulting in plasmidpIC2051 (Appendix 4). A FseI/Xba1 fragment containing the GFP ORFflanked by LoxA and LoxM sites in opposite orientation was subclonedfrom pIC2051 into the XbaI and FseI sites of pIC1951, resulting inplasmid pIC2121 (Appendix 5). pIC2121 contains a promoterless GFP codingsequence located between two heterospecific sites, replacing the coatprotein gene.

pIC1262 was made by cloning a 0.9 kb Ecl136II-Pst1 Arabidopsis actin2promoter fragment from pIC04 (actin2 promoter fragment cloned in aplasmid vector) into the Hind3-blunt and Pst1 sites of pIC08 (35Spromoter-LoxP-Cre-Nos terminator in pUC19). pIC1321 was made byreplacing the Nos terminator of pIC1262 by a DNA fragment containing aLoxM (in opposite orientation relatively to the LoxP site) site followedby the Ocs terminator. pIC1321 (Appendix 6) contains the followinginsert in pUC19: Arabidopsis actin2 promoter-LoxP-Cre Orf-LoxM-Ocsterminator.

Recombination of a Replicating Plasmid with a Non-Replicating PlasmidTarget Site (FIG. 3)

pIC2121 was co-bombarded with pIC1321 in wild-type N. benthamiana leaf,Phaseolus vulgaris leaf, and Phaseolus vulgaris bean cell suspensionculture. As a control, pIC2051 was co-bombarded with pIC1321 in the sameplant tissues. After three days, replication of the pIC2121 insert leadsto increased recombination with pIC1321 and results in exchange of theCre ORF with the GFP coding sequence. Fusion of GFP to the arabidopsisactin2 promoter leads to expression of GFP. No GPF expressing cells weredetected in the control experiment with non-replicating plasmid pIC2051(FIG. 3).

Example 3

In this example, we show that replication of a plasmid containing aninsert to be targeted can increase the frequency of recombination with atarget site stably inserted on a plant chromosome. Recombination ismediated by Cre recombinase and takes place at the loxP and LoxM sitespresent on both the replicating plasmid and the target site, and Cre isdelivered on a co-transformed plasmid (FIG. 4).Description of Plasmids:

An adaptor (made with primers adlox15 [tcg aga taa ctt cgt ata gca tacatt ata cga agt tat agc t] and adlox16 [ata act tcg tat aat gta tgc tatacg aag tta tc]) containing a LoxP site flanked with XhoI and SacI wascloned in pIC01 digested with XhoI and SacI. The resulting plasmid,pIC2745 contains the DNA fragment (35S promoter-LoxP-Gus-Ocs terminator)in pUC118. An adaptor (made with primers adlox17 [gat cat aac ttc gtataa tct ata cta tac gaa gtt att] and adlox18 [cta gaa taa ctt cgt atagta tag att ata cga agt tat]) containing a LoxM site (in oppositeorientation) flanked with BamHI and XbaI sites was cloned in pIC2745digested with BamHI and XbaI, resulting in plasmid pIC2755. AnEcoRI-Hind3 fragment was subcloned from pIC2755 into the EcoRI and Hind3sites of the binary vector pICBV1 (vector developed at Icon Genetics;any other binary vector system would be equally suitable for thiscloning). The resulting plasmid, pIC2764 (Appendix 7) contains theinsert (35S promoter-LoxP-Gus-LoxM-Ocs terminator) in a binary vector.

The Cre ORF was amplified by PCR from pIC08 with primers crerecomb1(CATGCCATGG CCAATTTACT GACCT) and crerecomb2 (TGCTCTAGAC TAATCGCCATCTTCCAGC) and cloned as a NcoI-XbaI blunt fragment into the PstI bluntand NcoI sites of pIC011. The resulting clone, pIC2721 (Appendix 8),contains the Cre ORF under the control of the Hbt promoter (chimericpromoter containing the 35S enhancer fused to the basal promoter of themaize C4PPDK gene; see Sheen, EMBO J., 1995, 12, 3497) in pUC18.

Increased Recombination of a Replicating Plasmid with a ChromosomalTarget Site.

Construct pIC2764 was introduced in Agrobacterium strain GV3101 byelectroporation, and the transformed bacteria used for Nicotianabenthamiana transformation. Thirty transgenic N. benthamiana plants werestained with an X-gluc solution (Jefferson, 1987, Plant Mol. Biol.Reporter, 5, 387-405) to select plants with high levels of Gusexpression. Plants expressing Gus were bombarded with a mix of plasmidspIC2721 and pIC2121. After delivery to plant cells, a DNAA genomecontaining GFP is expected to be released from pIC2121 and to replicate.Cre-mediated recombination results in exchange of the Gus codingsequence at the target locus on the chromosome by the GFP codingsequence, placing GFP under control of the 35S promoter. In a controlexperiment, pIC2051 (non-replicating promoterless GFP construct) wascobombarded on transgenic N. benthamiana plants expressing Gus. More GFPexpressing cells were detected when pIC2121 was cobombarded with pIC2721than in the control experiment.

pIC2764 was also transformed in a Phaseolus vulgaris cell suspensionculture line developed at Icon Genetics. Stably transformed colonieswere stained with X-Gluc to select lines with a high level of Gusactivity. Cells from two clones expressing Gus at high level weremultiplied and bombarded with a mix of plasmids pIC2121 and pIC2721 orwith a mix of plasmids pIC2051 and pIC2721. More GFP positive cells wereobserved a week after bombardment when pIC2121 was cobombarded withpIC2721 in comparison with the control experiment.

Example 4

In this example we show that site-targeted recombination events asdescribed in example 3 can lead to the production of stably transformedbean cells. In this example, the BAR gene (FIG. 5) is replaced by GFP,but the targeting strategy is identical as in example 3.Plasmid Description and Experiment

PIC2103 was made by cloning a SstI-BamHI fragment from pIC012 (Nospromoter-Bar coding sequence-Ocs terminator in pUC118) in the SstI-BamHIsites of pIC551. A pIC2103 FseI-XbaI fragment containing the BAR codingsequence flanked by two heterospecific Lox sites in oppositeorientations was subcloned in the FseI-XbaI sites of pIC1951 resultingin plasmid pIC2574 (Appendix 9). pIC2574 contains a promoterless BARcoding sequence cloned between two heterospecific sites, replacing thecoat protein gene.

pIC2574 was digested with BgI2 and religated. The resulting clonepIC2948 (Appendix 10) has a deletion of the Al1 (replicase), Al2 and Al3ORFs.

Cells from two P. vulgaris transgenic lines (stably transformed withpIC2764) described above were bombarded with a mix of plasmids pIC2574and pIC2721 or with a mix of plasmids pIC2948 and pIC2721. Transformedclones were selected on plates containing phosphinotricin (PPT).Transformed clones were analyzed by PCR to make sure that they had beenproduced by site-specific recombination. More PPT resistant clones wereobtained when pIC2574 was used than when the non-replicating controlpIC2948 was used.

Example 5

In this example, the strategy is similar to that of example 4. However,here, the replicase is present on a non-replicating plasmid with Cre(FIG. 6). The benefit of this approach is that replication of thereplicon is only transient and stops when the non-replicating plasmidcarrying the replicase disappears. The advantage is that transformedcells are free of replicating plasmid, are healthier and transgenicplants can be regenerated more easily.Plasmid Description and Experiment:

A fragment containing the Al1 (replicase), Al2 and Al3 ORFs wasamplified from plasmid pIC1664 using primers Al1xho1 (tct ctc gag ttacaa ata tgc cac cac ctc aaa g) and Al1xba1 (gct cta gag gat cta ttt ctatga ttc gat aac c). The amplified fragment was cloned as a Xho1 Xba1fragment in the Xho1 and Xba1 sites of pIC01 (35S promter-Gus codingsequence-Ocs terminator in pUC118). The resulting plasmid, pIC2821,contains the BGMV replicase under the control of the 35S promoter.

An adaptor (ecopst1, ecopst2) was cloned in the EcoRI site of pIC2721.The resulting clone, pIC2955, has the EcoRI site replaced by the Mfe1and Pst1 sites. Two fragments from pIC2821 (a EcoRI-NcoI fragment and aNcoI-Pst1 fragment) were cloned in a three-way ligation in pIC2955digested with Mfe1 and Pst1. The resulting plasmid, pIC2966 (Appendix11), contains the BGMV replicase expressed from the 35S promoter and theCre coding sequence under the control of the Hbt promoter.

Cells from two P. vulgaris transgenic lines (stably transformed withpIC2764) described above were bombarded with a mix of plasmids pIC2948and pIC2966 or with a mix of plasmids pIC2948 and pIC2721. Transformedclones were selected on plates containing phosphinotricin (PPT).Transformed clones were analyzed by PCR to check that they had beenproduced by site-specific recombination. More PPT resistant clones wereobtained when pIC2948 was replicating (due to the replicase on pIC2966)than in the non-replicating control when pIC2948 is cotransformed withpIC2721.

Example 6

In this example, a replicating clone carrying GFP is co-bombarded with areplicating Cre-expressing clone and the BGMV DNAB genome (FIG. 7). Allthree clones are able to replicate, and because of the presence of the Bgenome, all three clones are able to move to neighboring cells wherethey also replicate. The result is an increase in the number of cellswhere site-specific recombination events take place.Plasmid Description and Experiment

The Cre ORF was excised from pIC903 (Cre ORF cloned in pGem-T fromPromega) as a SacI blunt-Pst1fragment and cloned in the BamHI blunt andPst1 sites of pIC1664. The resulting plasmid pIC2736 (Appendix 12)contains the Cre coding sequence under the control of the BGMV coatprotein promoter in a DNAA replicating vector.

pIC2121 was co-bombarded with pIC2736 and pIC1914 (Nsi1-digested) inleaves of transgenic Nicotiana benthamiana plants transformed withpIC2764. In a control experiment, pIC2051 was co-bombarded with pIC2721.A week after bombardment, more GFP-expressing cells were detected in theexperiment than in the control.

Example 7

This experiment is similar to the one described in example 6, butdiffers by the inability of the B genome clone to replicate and to moveto neighboring cells (FIG. 8). The B genome clone is only transientlyexpressed and disappears after some time. It is advantageous toeliminate the B clone as it has been shown that expression of theBL1gene is in large part responsible for the disease symptoms ofgeminivirus-infected plants (Pascal et al., 1993, Plant Cell, 795-807).It has also been shown that transient expression of genes of the Bgenome is sufficient for systemic movement of DNAA genome for TGMV(Jeffrey et al., 1996, Virology, 223, 208-218).Plasmid Description and Experiment

An EcoRI-SacI fragment from pIC04 containing the Arabidopsis actin2promoter was cloned in the EcoRI and SacI sites of pIC02 (35Spromoter-Gus coding sequence-Ocs terminator in pUC118), resulting inplasmid pIC2779. A PCR fragment containing the BGMV BL1 Orf wasamplified from Phaseolus vulgaris DNA (extracted from BGMV infected leaftissue) using primers Bl1Xho1 (gcc tcg agc tta aat gga ttc tca gtt agc)and Bl1bam (cgg gat cct tat ttc aaa gac ttt ggt tga g). This fragmentwas cloned as an Xho1-BamHI fragment in pIC01, resulting in plasmid2781. A PCR fragment containing the BGMV BR1 ORF was amplified frompIC1914 DNA using primers Br1nsi1 (cga tgc atc aca cga att aat aat gtatgc gtc) and Br1bam (cgg gat cct tat cca aca taa tca aga tca aat g).This fragment was cloned as a Nsi1-BamHI fragment in pIC2779, resultingin plasmid 2792. Two pIC2781 fragments (EcoRI blunt-BamHI andBamHI-Hind3) were cloned in a three ways ligation in pIC2792 digestedwith Pst1 (blunted) and Hind3. The resulting plasmid, pIC2807 (Appendix13), contains the BR1 and BL1 ORFs under control of the Arabidopsisactin2 promoter and the 35S promoter (respectively), in pUC118.

pIC2121 was co-bombarded with pIC2807 and pIC2736 in leaves oftransgenic Nicotiana benthamiana plants transformed with pIC2764. In acontrol experiment, pIC2051 was co-bombarded with pIC2721. A week afterbombardment, more GFP-expressing cells were detected in the experimentthan in the control.

Example 8

This example is similar to example 6, but here the recombinase isexpressed from the target site instead of being delivered from areplicating clone (FIG. 9). The advantage is that Cre is alreadyexpressed in all the cells where replicating clones move. In addition,recombination at the target site displaces cre, preventing its furtherexpression.

Plasmid Description and Experiment

The actin2 promoter-LoxP-Cre Orf-Nos terminator fragment from pIC1321was subcloned as a Not1 blunt-SacI fragment into the SmaI and SacI sitesof the binary vector pBIN19, resulting in construct pIC1593 (Appendix14).

A LoxP-Gus-Ocs terminator-LoxP fragment was amplified from plasmid pIC02using primers LoxPgus (ggc atc gat ata act tcg tat agc ata cat tat acgaag tta tac aat ggg tca gtc cct tat g) and LoxPocs (gcc cat gga taa cttgct ata atg tat gct ata cga agt tat gtc aag gtt tga cct gca c). Theamplified fragment was digested with ClaI and NcoI and cloned in pIC591(pIC011 with BamHI site replaced by ClaI) digested with ClaI and NcoI.The resulting plasmid, pIC2553, contains the Gus gene flanked by LoxPsites inserted between the promoter and the coding sequence of GFP.

pIC1593 was introduced in Agrobacterium strain Agl1 by electroporationand transformed agrobacteria used to transformed Nicotiana benthamiana.DNA extracted from 10 transformants was used to test for the presence ofthe transgene by PCR. All plants were found positive when PCR wasperformed with primers for the kanamycin transformation marker or forthe Cre gene. To test functionality of the Cre recombinase in transgenicplants, one leaf of 25 transformants was bombarded with plasmid pIC2553.In presence of cre, recombination of the LoxP sites of pIC2553 resultsin expression of the GFP gene. Leaves of plants that were found toexpress Cre were bombarded with a mix of plasmids pIC2121 and pIC1914(NsiI-digested). In a control experiment, leaves of the same transgenicplants were bombarded with a mix of plasmids pIC2051 and pIC1914(Nsi1-digested). More GFP-expressing cells were observed in theexperiment than in the control.

Example 9

This example shows that replication of a plasmid containing a DNAsequence homologous to a target sequence in the genome can lead tohomologous recombination with this target sequence (FIG. 10).

Plasmid Description and Experiment

A fragment of the Phaseolus vulgaris ALS gene was amplified from genomicDNA using degenerate primers alsdpr1 (cgg gat ccc agg tgg ngc wtc matgga gat) and alsdpr2 (cgg agc tcg cat aca cag thc crt gca t) and wassequenced directly. Sequence information was used to design two primers(alspr3: cga cag cgt cgc cct cgt tgc cat c and alspr4: gat ggc aac gagggc gac gct gtc g) that overlap with a proline (equivalent to Pro-165 ofmaize AHAS108 [Lee et al., 1988, EMBO J., 7, 1241-1248]). Alspr3 andalspr4 contain a nucleotide substitution to change this proline toalanine. Using PCR, a AHAS DNA fragment with a proline mutated toalanine was amplified from bean genomic DNA using primers alsdpr1,alsdpr2, alspr3 and alspr4. This DNA was cloned in pIC2171 as aSacI-BamHI fragment, resulting in plasmid pIC2834 (Appendix 15).

As a control for a non-replicating plasmid, the SacI-BamHI fragment frompIC2834 was subcloned in pUC19, resulting in plasmid 2857 (Appendix 16).

Bean cell suspension cultures were prepared from Phaseolus vulgaris leaftissue. Sixty plates, each containing approximately 10⁶ cells, werebombarded with plasmid pIC2834. As a negative control, sixty plates werebombarded with plasmid pIC2857, and 40 additional plates were notbombarded but grown in the same conditions. The transformed cells wereplated on solid culture medium containing 20 ppb chlorosulfon (Glean,technical grade, Dupont). Putative events identified 4 to 6 weeks afterbombardment were selected on fresh media containing 50 ppb chlorosulfon.The resistant clones were analyzed by PCR amplification and sequencing.More resistant clones resulting from the expected change (Proline toAlanine at the targeted codon) were obtained in the experiment (usingthe replicating clone pIC2834) than in the controls.

Example 10

This example shows that replicating geminiviral clones can be deliveredby agroinfiltration.

Plasmid Description and Experiment

A binary vector containing the proreplicon part of pIC1694 was made bysubcloning a XhoI-NarI fragment from pIC1694 into pICBV11 digested withXho1 and Cla1. The resulting clone, pICH4300 (FIG. 11), contains the GFPgene under control of the BGMV coat protein promoter and the Al1/2/3genes, between duplicated CRs. pIC4300 was transformed in Agrobacteriumstrain GV3101. Agrobacterium cells were grown overnight in LB containing100 uM acetosyringone. The following day, bacteria were pelleted andresuspended at an OD of 0.8 in a solution containing 10 mM MES, 10 mMMgSO4 and 100 uM acetosyringone. Resuspended agrobacterium cells wereinfiltrated in Nicotiana benthamiana leaves. From two days postinoculation to more than 2 weeks, strong GFP fluorescence was observedin the infiltrated area. To check that geminiviral replicons wereformed, genomic DNA was extracted from infiltrated areas 3 days postinoculation, and analyzed by Southern blot. Undigested DNA analyzed witha GFP probe revealed the presence of nicked open circular DNA andsupercoiled DNA, while DNA linearized with BamHI gave a single migratingfragment. By comparison of the intensity of the signal of linearized DNAwith the signal of plasmid DNA of known concentration, it was estimatedthat replicons are present at 15 to 30000 copies per cell.

Example 11

In this example, we show that geminiviral clones lacking replicase canreplicate efficiently when the replicase is provided in trans.

Plasmid Description

A PCR product amplified from pICH4300 with crpr6 (cgc aat tgc tcg agcttt gag gtg gtg gca tat ttg) and gfppr1 (cgctgaacttgtggccgttcac) wascloned as a Xho1 BamHI fragment in the Xho1 BamHI sites of pICH4300. Theresulting clone pICH5184 is similar to pICH4300 but lacks a fragment ofthe AL1 gene located outside the proreplicon area in pICH5184. A GFPproreplicon lacking the replicase was made by cloning a PCR productamplified from pICH4300 with crpr9 (cgg tca tga ttc tca agc aca gta tggcat att tgt aaa tat gcg agt gtc) and crpr8 (gc tct aga gac acg tgg aggcgt acg g) in the BspH1 and Xba1 sites of pICH5184. Plasmid pICH5170(FIG. 11) was made by cloning an Xho1 Xba1 fragment (Al1/2/3 Orfs) ofpICH2821 in pICBV16 (Icon Genetics Binary vector, Kan selection).Al1/2/3 Orfs are under control of the 35S promoter in pICH5170. A PCRproduct amplified from pICH4300 with primers crpr8 (gc tct aga gac acgtgg agg cgt acg g) and crpr9 (cgg tca tga ttc tca agc aca gta tgg catatt tgt aaa tat gcg agt gtc) was cloned as a BspHI Xba1 fragment inpICH4300. The resulting plasmid, pICH5203 (FIG. 11), is similar topICH4300 but lack the Al1/2/3 Orfs. PICH4699 is similar to pICH4300 butthe GFP coding sequence was replaced by a DNA sequence containingLoxA-Gus coding sequence-nos terminator-LoxM in antisense orientation.

Experiment

pICH5203, pICH4699 and pICH5170 were transformed in Agrobacterium strainGV3101. Nicotiana benthamiana leaves were infiltrated as describedabove. pICH5203 was infiltrated alone, with pICH4699 or with pICH5170,and pICH4300 was infiltrated as a positive control. Genomic DNA wasextracted from infiltrated areas 4 days later and was analyzed bySouthern blotting with a common region probe. No replication wasdetected when pICH5203 was infiltrated alone (Lane 1-3, FIG. 11). ThepICH5203 replicon amplified at high level (FIG. 11, lane 4-6, fragmentb) in tissues coinfiltrated with pICH4699 (which replicatesconstitutively; amplified fragment (a) is shown on FIG. 11). It alsoreplicated efficiently when it was coinfiltrated with pICH5170 whichdoes not replicate (FIG. 11, lane 7-9), but at a lower level(approximately 5 times lower) than when pICH4300 was infiltrated alone(lane 10-12).

Example 12

In this example, recombination relies on the Streptomyces Phage PhiC31integrase system and recombination takes place between AttP and AttBsites. Site targeted transformation is performed using agrobacteriumtransformation but could also be performed by other means of delivery.Plasmid Description

pICH6272 (FIG. 12) consists of: the 34S promoter-AttB site-Gus codingsequence-Ocs terminator-AttB site (in inverse orientation)-GFP codingsequence-Nos terminator, cloned in pICBV10 (Icon Genetics Binary vectorwith Nos promoter-NptII coding sequence-Nos terminator for selection).The 34S promoter was cloned from pICP1159 (Chloramphenicol plasmidcontaining the 34S promoter-multicloning site-Mannopine synthaseterminator) as a Xba BgI2 fragment. The Gus coding sequence-Ocsterminator sequence block comes as a Sac1 Pst1 fragment from pIC01. TheGFP coding sequence-nos terminator comes from a pIC011-derived clonemissing the Pst1 site. The AttB recombination site (ccgcggtgcgggtgccagggcgtgcccttgggctccccgggcgcgtactccac) was synthesized fromoligonucleotides ordered from InVitrogen.

pICH7555 (FIG. 12) consists of: the arabidopsis actin 2 promoter-PhiC31integrase-Nos terminator-BGMV common region-AttP site-Bar codingsequence-Ocs terminator-35S promoter-AttP site in inverseorientation-BGMV common region, cloned in pICBV10. The common regioncomes from pIC1694, the Bar-Ocs terminator from pIC012, the 35S promoterfrom pIC01 and the Actin 2-PhiC31 integrase-Nos terminator block frompICP1010. The AttP recombination site(gtagtgccccaactggggtaacctttgagttctctcagttgggggcgta) was synthesized fromoligonucleotides ordered from InVitrogen.

Experiment

Plasmid pICH6272 was stably transformed in Nicotiana tabacum byAgrobacterium transformation. Transgenic plants were checked for Gusexpression by staining leaf tissue with X-Gluc. Two transformantsexpressing Gus were chosen to be used for site-targeted transformation.Recombination at the AttP sites on the replicons (derived from pICH7555)with the AttB sites at the target site (derived from pICH6272) shouldplace the promoterless BAR gene (from the replicon) under control of the34S promoter, thereby conferring PPT resistance to transformed cells.Leaf discs of both transformants were inoculated with Agrobacteriacarrying plasmid pICH7555 or by a mixture of Agrobacterium culturescontaining pICH7555 and pICH5170. In the presence of PhiC31 integrase,site-specific recombination of the two AttP sites on the replicon cantake place at either one of the two AttB sites at the target locus. Whenthe first AttP site of construct pICH7555 or of the pICH7555-derivedreplicon recombines with the first AttB site at the target locus, thepromotorless Bar gene from pICH7555 or from the replicon is placed undercontrol of the 34S promoter at the target site. Selection fortransformants was made on PPT-containing media. More transformants wereobtained when the gene to be targeted replicated transiently (transientexpression of both pICH7555 and pICH5170) than when it did not replicate(pICH7555 alone). Transformants were analyzed by PCR and Southernblotting to confirm that they were site-targeted transformants.

Example 13

In this example, the target site and the gene to be targeted (present ona proreplicon) are first transformed in separate plants. Delivery of thegene to be targeted is achieved by hybridization.

Plasmid Description:

-   -   pICH6313 (FIG. 12) is derived from plasmid pICH6272. A BgI2 SpeI        fragment was subcloned from pICH6303 (Al1/2/3 Orfs linked to an        Internal Ribosome Entry Site in binary vector) into the Xba1        BamHI sites of pICH6272. The resulting plasmid contains the Gus        and Al1/2/3 genes under control of the 34S promoter. pICH6040        (FIG. 12) consists of: the 35S promoter-AttB site-Bar coding        sequence-Ocs terminator AttP site-GFP coding sequence-Nos        terminator in pUC118. The 35S promoter sequence comes from        pIC01, the Bar-Ocs terminator from pIC012 and the GFP-Nos        terminator from pIC011. pICH6040 was designed as a test        construct to check PhiC31 expression in transgenic plants: upon        expression of PhiC31 integrase, intramolecular recombination of        AttB with AttP leads to fusion of the GFP coding sequence to the        35S promoter, and to GFP expression.        Experiment

pICH7555, pICH6313 and pICH6272 were stably transformed into Nicotianatabacum using agrobacterium transformation. pICH6313 transformants thatexpressed Gus were selected to be used in crosses with pICH7555transformants. These transformants are also expected to express the BGMVreplicase as it is linked to Gus by an IRES. pICH7555 transformants werechecked for the presence of the proreplicon by PCR, and for activity ofthe PhiC31 integrase by bombardment of leaf tissue with test constructpICH6040. pICH6313 transformants were crossed as female with pICH7555transformants. In F1 plants, expression of the Al1/2/3 genes frompICH6313 results in formation of replicons from the pICH7555 transgene.Recombination of replicon molecules with the target site results infusion of the BAR gene to the 34S promoter. At the same time,replacement of the Gus coding sequence-IRES-Al1/2/3 Orfs by the Barcoding sequence-Ocs terminator-35S promoter results in termination ofreplication of the replicon. In control crosses (no replication)pICH6272 transformants were crossed as female to pICH7555 transformants.F1 plants from both types of crosses were grown without selection, andBasta selection was applied on F2 seedlings. More Basta resistant plantswere obtained from crosses with pICH6313 than in crosses with pICH6272.Basta resistant plants were checked by PCR and Southern blot analysis toconfirm that they resulted from site-targeted recombination events.

Example 14

In this example we use an RNA virus provector system as an assay todetect successful site-targeted DNA recombination events. Recombinationevents at LoxP sites on separate fragments of a provector system lead toDNA molecules that are transcribed into functional viral transcriptscapable of amplification. Using this assay, we show that replication ofa DNA sequence increases the rate of site-specific recombination with anon-replicating target sequence.Plasmid Description

pICH4371 consists of a 5′ provector based on the TVCV RNA virus.pICH4371 contains the arabidopsis actin 2 promoter—the TVCV polymerase—atruncated version of the movement protein—a LoxP site-Nos terminator, inbinary vector. pICH4461 consists of the 3′ end provector. It contains aLoxP site-GFP coding sequence-viral 3′ NTR-Nos terminator, in binaryvector. pICH7311 was made by cloning a EcoRI-PstI fragment from pICH4461(containing the 3′ provector fragment) into pICH6970 (LoxA-Bar codingsequence-LoxM between 2 BGMV common regions in Binary vector) digestedwith EcoRI-PstI. pICH7311 consists of LoxP-GFP coding sequence-TVCV 3′NTR-Nos terminator between two BGMV common regions in binary vector(FIG. 13). pICH1754 consists of: Arabidopsis actin 2 promoter-LoxP-crecoding sequence-LoxM-Ocs terminator cloned in pICBV10. pIC1754 is usedhere to provide cre recombinase.

Experiment:

-   -   pICH4371, pICH7311, pICH5170 and pICH1754 were transformed into        Agrobacterium strain GV3101. pICH4371 was coinfiltrated in N.        benthamiana leaves with pICH7311, pICH1754, with or without        pICH5170 (BGMV replicase). Infiltration with pICH5170 resulted        in more GFP sectors than without pICH5170 (FIG. 13) showing that        amplification of 3′-end provector results in an increase of        site-specific recombination events. As a negative control,        pICH7311 was infiltrated with or without pICH5170. No GFP        expression could be detected in either case indicating that GFP        is not expressed from the 3′ provector clone alone. Also,        pICH4371 was coinfiltrated with pICH4461 and pICH1754. The same        number of recombination events was observed as when pICH4371 was        coinfiltrated with pICH7311 and pICH1754 (not shown).

Example 15

In this example we show that replication of a DNA sequence increases therate of homologous recombination with a non-replicating homologoussequence. Recombination events are detected by mutating a non-functionalRNA proreplicon to a functional one, leading to amplification and toGFP-expressing leaf cell sectors.Plasmid Description

Plasmid pICH7477 (FIG. 14) was made by ligating three fragments frompICH4351; a KpnI SphI-blunt fragment, a SphI-blunt Xho1 fragment and aXho1 Kpn1 fragment. The resulting clone, pICH7477, contains a frameshiftin the TVCV RNA dependent RNA polymerase (Rdrp) Orf at the Sph1 site,and is therefore a non-functional proreplicon clone. A non-mutantfragment from the TVCV Rdrp Orf was PCR-amplified from pICH4351 withprimers rdrppr3 (ttt ccatgg att acc ctg tta tcc cta aag gca tct cgt cgcgtt tac) and rdrppr4 (ttt ctgcag gaa atg aaa ggc cgc gaa aca ag) andcloned as a Nco1 Pst1 fragment in pICH7423 (pICH7423 is a derivative ofpICH1694 in which a Hind3 Nco1 from the coat protein promoter region wasremoved). The resulting clone, pICH7480 (FIG. 14), contains anon-mutated fragment of TVCV flanked on one side by a I-SceI restrictionsite, in a geminiviral proreplicon. The Nco1 Pst1 fragment from pICH7480was subcloned in Nco1 and Pst1 sites of pICH6970. The resulting clone,pICH7499 (FIG. 13), is similar to pICH7480 but cannot replicateautonomously due to the lack of replicase. It can however replicate whenthe replicase is provided in trans.

Experiment:

Plasmid pICH4351 is a proreplicon carrying GFP that is based on the RNAvirus TVCV. In pICH7477, replicons cannot be produced due to aframeshift in the ORF of TVCV. Nicotiana benthamiana plants wereinfiltrated with agrobacterium containing plasmid pICH7480. As anonreplicating control, pICH7499 was agroinfiltrated in a second plant.One day later, both plants were infiltrated with pICH7477 and pICH7500(35S promoter-I-SceI endonuclease-Nos terminator). Expression ofpICH7500 leads to I-SceI restriction endonuclease and cleavage ofgeminiviral replicons at the I-SceI restriction site. Homologousrecombination of the linearized fragments with the mutated part of theTVCV Orf leads to restoration of functional TVCV proreplicons. More GFPexpressing sectors were formed with pICH7480 than with pICH7499.

In an variation of this experiment, the replicase for the geminiviralreplicons is expressed in trans transiently. Nicotiana benthamianaplants were infiltrated with pICH7480 alone or with pICH7480 andpICH5170. One day later, all plants were infiltrated with pICH7477 andpICH7500. More GFP sectors were obtained in plants inoculated withpICH5170 than in plants inoculated with pICH7480 alone.

In another experiment, pICH7477 was stably transformed in N.benthamiana. Transformants were infiltrated with pICH7480 or pICH7499.One day later, I-SceI restriction endonuclease was delivered byinfiltrating the same areas with pICH7500. More GFP sectors wereobtained in plants infiltrated with pICH7480 than in plants infiltratedwith pICH7499.

In another experiment, transgenic plants for pICH7477 were infiltratedwith pICH7499 alone or with pICH5170. One day later Restrictionendonuclease was delivered by infiltrating the same areas with pICH7500.More GFP sectors were obtained in plants infiltrated with pICH5170 thanin plants infiltrated with pICH7499 alone.

1. A process of causing a targeted integration of DNA of interest into aplant cell nuclear genome, comprising: (i) providing plant cells with anamplification vector, or a precursor thereof, capable of replication inplant cells, said vector comprising: (a) DNA sequence(s) encoding ageminiviral origin of replication and functional in plant cells, (b) DNAsequence(s) necessary for site-specific and/or homologous recombinationbetween the vector and a host nuclear DNA, and (c) optionally, furtherDNA of interest; (ii) providing said plant cells with a replicase geneof the replicase involved in replicating the amplification vector on anon-replicating vector, whereby the replication of said amplificationvector in said plant cells is transient; (iii) optionally providingconditions that facilitate vector amplification and/or cell to cellmovement and/or site-specific and/or homologous recombination; and (iv)selecting cells having undergone recombination at a predetermined sitein the plant nuclear DNA.
 2. A process of causing a targeted integrationof DNA of interest into a plant cell nuclear genome, comprising thefollowing steps: (i) transfecting or transforming a plant cell with afirst DNA comprising a sequence which, when integrated in the plant cellgenome, provides a target site for site-specific and/or homologousrecombination; (ii) selecting a cell which contains said target site forsite-specific and/or homologous recombination in its nuclear genome;(iii) transfecting or transforming said selected cell with a second DNAcomprising a region for recombination with said target site and a firstsequence of interest; (iv) optionally providing enzymes forrecombination; and (v) selecting cells which contain the sequence ofinterest from the second DNA integrated at the target site, whereby atleast one of said first or said second DNA is delivered by anamplification vector, or a precursor thereof, capable of replication ina plant cell and comprising (a) DNA sequence(s) encoding a geminiviralorigin of replication functional in the plant cell, and wherein thereplicase gene of the replicase involved in replicating theamplification vector is provided to said plant cell on a non-replicatingvector whereby the replication of said amplification vector in saidplant cell is transient.
 3. The process according to claim 1 or 2,wherein said amplification vector comprises DNA sequence(s) necessaryfor homologous recombination.
 4. The process according to claim 1 or 2,wherein said providing a plant cell with an amplification vector, or aprecursor thereof, or said transfecting or transforming is done byAgrobacterium-mediated delivery.
 5. The process according to claim 1 or2, wherein said providing a plant cell with an amplification vector, ora precursor thereof, or said transfecting or transforming is done bydirect viral transfection.
 6. The process according to claim 1 or 2,wherein said providing a plant cell with an amplification vector, or aprecursor thereof, or said transfecting or transforming is done bynon-biological delivery.
 7. The process according to claim 1 or 2,wherein said providing a plant cell with an amplification vector or saidtransfecting or transforming is done by conversion of a vector or apro-vector DNA that was pre-integrated into a plant nuclear DNA to forman autonomously replicating plasmid.
 8. The process according to claim 1or 2, wherein said amplification vector is released from a precursorthereof which has two origins of replication.
 9. The process accordingto claim 1 or 2, wherein said amplification vector is a DNAvirus-derived vector.
 10. The process according to claim 1 or 2, whereinsaid amplification vector is a DNA copy or a replication intermediate ofan RNA virus-derived vector.
 11. The process according to claim 1 or 2,wherein said amplification vector is of retrotransposon origin.
 12. Theprocess according to claim 1 or 2, wherein the amplification vector hasadditionally viral functions selected from the group consisting of:functions for reverse transcription, host infectivity, cell-to-celland/or systemic movement, integration into a host chromosome, viralparticle assembly, control of silencing by host, and control of hostphysiology.
 13. The process according to claim 1 or 2, wherein saidhomologous or site-specific recombination is one-sided.
 14. The processaccording to claim 1 or 2, wherein said homologous or site-specificrecombination is two-sided.
 15. The process according to claim 1 or 2,wherein said site-specific recombination is promoted or facilitated byrecombination enzymes selected from the group consisting of: sitespecific recombinases, restriction enzymes, integrases, and resolvases.16. The process according to claim 1 or 2, wherein said homologousrecombination is promoted or facilitated by recombination enzymesselected from the group consisting of: RecA-like proteins, rare-cuttingendonuclease of HO type, and I-Scel endonuclease.
 17. The processaccording to claim 1 or 2, wherein said amplification vector isassembled in a process of recombination.
 18. The process according toclaim 2, wherein said first DNA contains a selectable marker for theselection of step (ii).
 19. The process according to claim 2, whereinsaid first DNA comprises additionally a second sequence of interest. 20.The process according to claim 2, wherein the recombination of saidfirst and said second DNA establishes a functional sequence.
 21. Theprocess according to claim 2, wherein said first and said second DNAeach additionally contains a fragment of a selection marker, which makesa selection marker as a result of said recombination.
 22. The processaccording to claim 2, wherein said second DNA is delivered by anamplification vector or a precursor thereof.
 23. The process accordingto claim 2, wherein the function of a sequence introduced into the plantin steps (i) and (ii) is destroyed in steps (III) to (v).
 24. Theprocess according to claim 1 or 2, wherein expression of genes involvedin non-homologous recombination is inhibited or suppressed.
 25. Theprocess according to claim 1 or 2, wherein the end result ofrecombination is a site-directed mutation.
 26. The process according toclaim 1 or 2, wherein said geminivirus is bean golden mosaic virus(BGMV).